Neural stem cells (NSC) have tremendous therapeutic potential in the repair of central nervous system injury and disease. Knowledge of NSC biology will also aid in the understanding of developmental brain disorders as well as brain tumors, which may result from abnormal NSC proliferation. A fundamental property of neural and other stem cells is their ability to undergo self-renewing proliferation. This application focuses on NSC proliferation and self-renewal and is based on our previous discovery-driven work, identifying genes enriched in neural progenitors compared to their more differentiated progeny. It is hypothesized that genes expressed in multiple stem cell populations will play important roles in NSC self-renewal. The first two aims of this proposal focus upon three genes that share the characteristics of restricted expression in CNS germinal zones and expression in multiple stem cell-containing cultures: MELK (maternal embryonic leucine zipper kinase), PSP (phosphsoserine phosphatase), and TOPK (TLAK cell originating protein kinase). Expression analysis will be performed to determine whether these genes are synthesized by multipotent, self-renewing stem cells. This will consist of standard localization methods along with study of gene-specific, promoter-driven expression of enhanced green fluorescence protein. Progenitor cells transfected with these constructs will be sorted using FACS and assayed for their ability to serve as self-renewing, multipotent stem cells. The hypothesis that MELK, TOPK and PSP regulate neural stem/progenitor self-renewal will be directly tested by determining whether knockdown and/or over expression influences self-renewal of primary progenitors. The cell cycle mechanisms underlying any changes observed will be determined using a combination of FACS analysis of DNA content, and assay of cell cycle regulatory proteins. In addition to the study of individual genes, further experiments will test broader hypotheses with respect to gene expression in self-renewing NSCs. First, the hypothesis that genes expressed in multiple stem cell populations will regulate neural stem/progenitor cell proliferation will be more generally tested using 38 genes identified in a previous study. These genes will be further stratified and then screened for function in stem cell self-renewal using primary CNS progenitors. PTEN-deficient NSC have a greater capacity for self-renewal and genes enriched in these cells will be candidates to play important roles in NSC self-renewal. In another set of experiments, microarray analysis will be used to compare gene expression in PTEN-deficient and wild type neurosphere cultures to discover genes and gene networks regulating self-renewal.